ANAEROBIC TREATMENT OF ATRAZINE BEARING WASTEWATERS

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1 ANAEROBIC TREATMENT OF ATRAZINE BEARING WASTEWATERS Key Words: atrazine, anaerobic, pesticide, biodegradation P. K. Ghosh, Ligy Philip*, and M. Bandyopadhyay Department of Civil Engineering, Indian Institute of Technology, Kharagpur, W. Bengal, (INDIA), PIN Tel: , Fax: *Corresponding Author: ABSTRACT In the present study, performance of mixed microbial anaerobic culture in treating synthetic strong wastewater containing different concentration of atrazine was evaluated (R-1). Studies were also conducted in two hybrid reactors (A-1 and A-2) with wood charcoal as adsorbing matrix along with microbes. All the reactors for the treatment of atrazine bearing wastewater were operated in sequential mode with a Hydraulic Retention Time (HRT) of 5 days. In all the cases, Chemical Oxygen Demand (COD) removal was found to be above 82% within 5 days with an initial COD of above 1000 mg/l. Atrazine reduction was 43.8% and 40% with an initial concentration of and 1.0 mg/l respectively in R-1 after 5 days. Maximum atrazine reduction of 33.4% was observed in the same reactor with an initial atrazine concentration of 2.0 mg/l. Acetoclastic methanogenic activity test showed no inhibitory effect of atrazine up to a concentration of 2 mg/l, on methane producing bacteria. The performance of anaerobic microorganisms in removing atrazine in absence of any external carbon source (R-3) and in absence of external carbon and inorganic nitrogen source (R-4) was studied. With an initial atrazine concentration of 1.0 mg/l, R-3 could achieve 61.8% of atrazine removal in 34 days, whereas R-4 could affect only 44.2% of atrazine after 149 days. No further reduction of atrazine was observed on R-3 with time. INTRODUCTION Atrazine (2-chloro-4-ethylamino-4-isopropylamino-s-triazine), a chlorinated s-triazine group of herbicide, is a common contaminant in ground and surface water bodies (Muir and Baker, 1978; Belluck et al., 1991; Thurman et al., 1992). Many methods are in use for the removal of atrazine, like chemical oxidation, adsorption, hydraulic destruction, volatilization, phytoremediation, incineration and biodegradation. Each method has its own limitation. Although biodegradation of atrazine is a slow process, this is the only method by which atrazine can be mineralized. Mineralization of atrazine was observed by mixing several pure cultures but not by any of the 200 individual bacterial isolate (Mandelbaum et al., 1993). Although, atrazine degradation by pure culture microorganisms in aerobic (Cook and Hütter, 1981; Behki and Khan, 1986; Cook, 1987; Behki et al., 1993; Mandelbaum et al, 1993; Radosevich et al., 1995) and in absence of oxygen (Jessee et al., 1983; Crawford et al., 1998; Shapier et al., 1998) had been reported in literature, studies on the biodegradation of atrazine by anaerobic mixed microbial culture is scanty. Chung et al., (1996) studied the biodegradation of atrazine by anaerobic mixed microbial culture and no well known metabolites except hydroxyatrazine had been observed, which is non-phytotoxic. But, pure aerobic culture converted atrazine to dealkylated

2 metabolites, which are toxic to the same degree as atrazine (Kaufman and Blake, 1970). Also, it had been observed that anaerobic process was more suitable for the microbial degradation of BHC (Sidderamappa and Sethunathan, 1975) and DDT (Burge, 1971). There are many reported cases where atrazine was mixed with domestic wastewater. Atrazine from a manufacturing plant or from a distribution site when mixed with sewage water contained high organic carbon along with high concentration of atrazine. The main objective of the present study was to investigate the possibilities of bioremediation of atrazine bearing wastewater using anaerobic bacterial consortium and to develop a hybrid reactor for the same purpose. MATERIALS AND METHODS Feed Composition: Composition of feed is shown in Table 1. Analysis: Atrazine of technical grade was supplied by Rallis India Limited, Bombay (India). Dichloromethane, which was used as solvent, and all other chemicals used for feed preparation were of AR grade. COD, Mixed Liquor Suspended Solids (MLSS), and Mixed Liquor Volatile Suspended Solids (MLVSS) were determined as per standard methods (Standard Methods for the Examination of Water and Wastewater, 1995). COD TABLE-I Nutrient (S-1) COMPOSITION OF FEED COMPONENT CONCENTRATION (mg/l) K2HPO4 150 KH2PO4 MgCl2.6H2O CaCl2.2H2O NH4Cl FeCl2.4H2O ZnCl2 NiCl2.6H2O CoCl2.6H2O MnCl2.4H2O Yeast extract Dextrose (S-2) Dextrose 1000 Atrazine (S-3) Atrazine Variable (, 1.0 and 2.0)

3 was determined by closed refluxed method. ph measurement was done in a digital ph meter (DPH 500, SICO, India). Methane and total gas production was measured by water displacement method. Methane gas was measured, after scrubbing the total gas through KOH solution. VFA and Alkalinity determination was conducted as suggested by Dilallo and Albertson (1961). Specific Acetoclastic Methanogenic (SAM) activity test was conducted as described by Valcke and Vestrate (1983). Atrazine was extracted from wastewater sample by liquid-liquid extraction method using dichloromethane as extractant. Absorbance was measured in UV spectrophotometer using dichloromethane as blank. Maximum absorbance was observed at nm. The extraction efficiency was 88-92% in that method. Anaerobic microorganism: Anaerobic bacteria from an anaerobic bio-reactor from Environmental Engineering Laboratory, Indian Institute of Technology (IIT), Kharagpur, mixed with raw cow dung, was used as the seed sludge for the present study. Reactors used: The feed condition, working volume and the matrix used in different reactors are summarized in the Table-2. Table -II FEED CONDITION AND WORKING VOLUME OF THE REACTORS Reactor Feed Condition Working Matrix Vol. (ml) R-1 Nutrient (S-1) + Dextrose (S-2) + Atrazine (S- 3) 4000 X R-2 Nutrient (S-1) + Dextrose (S-2) 4000 X R-3 Nutrient (S-1) + Atrazine (S-3) 3500 X R-4 Nutrient (S-1) + Atrazine (S-3) - NH4Cl 350 X A-1 Nutrient (S-1) + Dextrose (S-2) + Atrazine (S- 3) A-2 Nutrient (S-1) + Dextrose (S-2) + Atrazine (S- 3) 800 X + W (10 g/l) 800 X + W (40 g/l) X=Microbial mass W=Wood charcoal All the reactors except R-3 and R-4 were operated in sequential mode with 5 day Hydraulic Retention Time (HRT) and instant feeding. Reactors R-3 and R-4 were operated in batch mode. Mixing was done manually. Studies were conducted at a constant temperature of 35 ±1OC in a temperature controlled chamber. Microbes in all the reactors were in suspended form.

4 RESULTS AND DISCUSSIONS COD and atrazine removal efficiency: COD removal from reactor R-1, after 5 days, with an initial atrazine concentration of mg/l, 1.0 mg/l and 2.0 mg/l was observed to be 85%, 82.2% and 84% respectively. These showed almost no inhibition effect of atrazine on anaerobic microorganisms. In the above three cycles, the initial COD concentration was1100 mg/l, 1120 mg/l and 1120 mg/l, whereas microbial mass concentration measured as MLSS was 2065 mg/l, 2095 mg/l and 2140 mg/l respectively. Atrazine removal from R-1, after 5 days, with an initial concentration of mg/l, 1.0 mg/l and 2.0 mg/l was observed to be 43.8%, 40% and 33.2% respectively. Maximum atrazine removal from R-1 with an initial concentration of 2 mg/l was observed to be 33.4% after 3 days. From reactor R-2, which did not contain atrazine but identical to R- 1 in all respects, COD removal was observed to be 86% (Figure-1). This showed no significant inhibitory effect of atrazine on the microorganisms even at a concentration of 2.0 mg/l of atrazine. With an initial atrazine

5 Concentration of 2.0 mg/l and MLSS concentration of 4235 mg/l atrazine removal of 35.8% was observed after 5 days. This shows that even about 2-fold increase in biomass concentration, atrazine removal did not increase significantly (Figure-2). Reactors A-1 and A-2 were operated in sequential mode with HRT of 5 days with an initial atrazine concentration of 2.0 mg/l. Maximum atrazine removal in reactor A-1 and A-2 was observed to be 64% and 69.4% after 3 days whereas removal was 37.1% and 38.5% after 10 days. MLSS in both the reactors was almost same i.e mg/l. Slight better performance of A-2 than A-1 in removing atrazine may be due to the higher adsorbent dose. Atrazine removal in A-1 and A-2 increased rapidly for first 3 days due to the adsorption of atrazine in wood charcoal and the decrease in performance may be due to the exhaust of adsorbent. In R-3, maximum atrazine removal of 61.8% was observed after 34 days of incubation. MLSS concentration in this reactor was 4845 mg/l. In R-4, an atrazine removal of 42% was observed after 150 days. MLSS concentration in the reactor was 4200 mg/l. These result showed that atrazine can be degraded by mixed microbial anaerobic culture in the absence of any external carbon and/or inorganic nitrogen source. Gas production: Total and methane gas production in R-1 and R-2 during first and second cycles of operation is shown in Figure-4. No significant difference in gas production was observed due to the presence of atrazine.

6 Methanogenic activity test: As methane producing bacteria is the most sensitive organism in the anaerobic microbial consortia, methanogenic activity test carried out. Specific Acetoclastic Methanogenic (SAM) activity test was conducted with the sludge of reactor R-1 after second cycle of operation to observe the effect of atrazine on methanogens. Same test was conducted with the sludge in reactor R-2 for comparison. No inhibition of atrazine on methane producing bacteria was observed. SAM for R-1 was 45.2ml methane/gmvss.day when fed with 2.0 mg/l of atrazine, where as for R-2 it was 52.8ml methane/gmvss.day in absence of atrazine. CONCLUSIONS Anaerobic mixed culture was able to remove atrazine and organic matter from atrazine bearing wastewaters. In the cycles, although COD removal efficiency was above 82%, atrazine removal was below 45%. In absence of any external carbon source, atrazine removal efficiency as high as 61.8% was observed whereas in absence of both, nitrogen and carbon source, maximum of 42% removal was observed. No adverse effect of atrazine at a concentration of 2.0 mg/l was observed on methanogens. REFERENCES 1. Behki et al., Appl. Environ. Microbiol., 59: (1993). 2. Behki, R. M. and Khan S. U., J. Agric. Food Chem., 34: (1986). 3. Belluck et al., American Chemical Society, Washington, D.C. (1991). 4. Burge, W.D., J.Agric. Fd. Chem. 19: (1971).

7 5. Chung et al.,wat. Res. 30: (1996) 6. Cook, A. M. and Hütter R. J., J. Agric. Food Chem. 29: (1981) 7. Cook, A. M., FEMS Microbiol. Rev. 46: (1987) 8. Crowford et al., Appl. Microbiol. Biotechnol., 49: (1998). 9. Jessee, J. A., Benoit R. A., Hendricks A. C., Allen G. C. and Neal J. L., Appl. Environ. Microbiol. 45: (1983). 10. Mandelbaum et al., Appl. And Environ. Microbiol. 59: (1993). 11. Muir, D. C. and Baker B. E., Weed Res., 18: (1978). 12. Radosevich et al., J. Env. Sci. Health, B30: 457 (1995). 13. Sidderamappa, R. and Sethunathan, N., Pestic. Sci. 6: (1975). 14. Standard methods for the examination of water and wastewater (1995). 15. Thurman et al., Env. Sci. Technol. 26: (1992). 16. U.S. Department of Health and Humane services. National Institute for occupational safety and health. (1991). Registry of toxic effects of Chemical Substances. Cincinnati, OH. 17. USEPA, Valke, D. and Vestrate, W., JWPCF, 55: 1191, (1983). 19. Weisenburger D., Amer. J. D., Ind. Med. 18: (1990).

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